US6185035B1 - Optical microscope - Google Patents

Optical microscope Download PDF

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Publication number
US6185035B1
US6185035B1 US09/257,154 US25715499A US6185035B1 US 6185035 B1 US6185035 B1 US 6185035B1 US 25715499 A US25715499 A US 25715499A US 6185035 B1 US6185035 B1 US 6185035B1
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Prior art keywords
sample
objective lens
light
microscope
disk
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US09/257,154
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English (en)
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Shinya Otsuki
Takeo Tanaami
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Yokogawa Electric Corp
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Yokogawa Electric Corp
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Assigned to YOKOGAWA ELECTRIC CORPORATION A JAPANESE CORPORATION reassignment YOKOGAWA ELECTRIC CORPORATION A JAPANESE CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: OTSUKI, SHINYA, TANAAMI, TAKEO
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G02B21/082—Condensers for incident illumination only
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/28—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00 for polarising
    • G02B27/281—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00 for polarising used for attenuating light intensity, e.g. comprising rotatable polarising elements

Definitions

  • This invention relates to an optical microscope; and more particularly, to an improvement thereof which results in reduced background light and achieves high S/N ratio in scanning type or reflecting type confocal optical microscopes.
  • FIG. 1 shows an example of a conventional confocal optical microscope, wherein light beam emitted from a suitable light source l, such as a mercury arc lamp or laser, is polarized with a polarizer 2 into a linearly polarized light beam.
  • a suitable light source l such as a mercury arc lamp or laser
  • the polarized light beam is transmitted through beam splitter 3 and is made incident to pinhole array disk 4 , such as a Nipkow disk.
  • the light beam that is transmitted through pinhole array disk 4 is polarized into a circularly polarized light beam by a quarter wavelength plate 6 (also abbreviated ⁇ /4 plate).
  • the circularly polarized light beam is focused by objective lens 7 and irradiated on sample 8 .
  • a plurality of spots irradiated on sample 8 is sequentially scanned with the light beam polarized circularly by rotating pinhole array disk 4 by driving the disk 4 with motor 5 .
  • the light beam reflected by sample 8 is focused by objective lens 7 and is linearly polarized by passing through the quarter wavelength plate 6 , and forms an image on the same pinhole array disk 4 .
  • the light beam that is passed through the pinholes of disk 4 is made incident to beam splitter 3 , reflected in the perpendicular direction, is made incident to analyzer 9 , and forms an image on camera 11 via relay lens 10 after being polarized linearly.
  • the image of sample 8 captured with camera 11 is displayed on the screen of monitor 12 .
  • the objective lens 7 has a reflectivity ranging from 0.5 to several percent.
  • Such reflection scarcely causes the above problem if an object whose surface reflectivity is high, such as a metallic surface, is to be measured.
  • a sample is to be measured whose surface reflectivity is low, such as the inside of multiple layers, skin, or the inside of a living body, for example having a reflectivity lower than 0.1%, such reflection as background light significantly hinders observation and measurement.
  • an object of the invention is to overcome the aforementioned and other deficiencies, disadvantages, and problems of the prior art.
  • Another object is to realize an optical microscope that has less background light and a high S/N ratio.
  • the invention encompasses an optical microscope that can observe the image of a sample by scanning the sample with light, and comprising a beam splitter branching a linearly polarized light beam and a light beam reflected from the sample; an objective lens for illuminating the sample by focusing the incident light beam on the sample; and a quarter wavelength plate disposed between the objective lens and the sample; wherein the S/N ratio of the microscope is improved and samples of low reflectivity can be readily measured by locating the quarter wavelength plate under the objective lens, that is on the sample side.
  • FIG. 1 is a drawing depicting a conventional optical microscope.
  • FIG. 2 is a drawing depicting an illustrative embodiment of the invention .
  • FIG. 3 is a drawing depicting use of a cover glass as the quarter wavelength plate.
  • FIG. 4 is a drawing depicting a quarter wavelength plate incorporated in an objective lens.
  • Mirrors 22 , 25 and 26 are provided to change the direction of a light beam.
  • a laser light beam is introduced into mirror 22 via FC connector 21 .
  • the laser light beam is introduced into FC connector 21 through an optical fiber, not shown, from a laser source, not shown.
  • An arc light source or a lamp may be used in place of the laser as a light source.
  • a light source may be located directly in place of the FC connector and not be limited to the light source introduction method using an optical fiber connected to the FC connector.
  • Collimating lens 23 is a lens for changing the reflected light from mirror 22 into parallel light beam.
  • ND filter 24 is a beam attenuating filter used for suitably controlling the quantity of light to be irradiated on sample 8 .
  • Collimating lens 23 and ND filter 24 are arranged between mirrors 22 and 25 .
  • polarizer 2 is located between mirrors 25 and 26 .
  • Micro lens disk 27 (in FIG. 2, the left half of the micro lens arrangement is omitted for clarity of description) is mounted above and in parallel with a pinhole array disk 4 in a manner as though polarized beam splitter 3 were sandwiched by microlens disk 27 and pinhole array disk 4 .
  • Pinhole array disk 4 is a disk in which a plurality of very small apertures are located in a predetermined pattern (This type of disk is called a Nipkow disk).
  • Micro lenses arranged in the same pattern as that of the pinholes in the pinhole array disk 4 are mounted on micro lens disk 27 .
  • the micro lens has the effect of enhancing the light utilization factor.
  • micro lens disk 27 and pinhole array disk 4 Approximately 20,000 micolenses and pinholes are arranged in micro lens disk 27 and pinhole array disk 4 , respectively, and forming helical patterns.
  • Laser light beam from mirror 26 illuminates an area, for example, of 10 mm by 7 mm on pinhole array disk 4 .
  • the two disks 27 and 4 are constructed to be concurrently rotated by a motor, not shown.
  • Tube lens 28 , objective lens 7 and quarter wavelength plate 6 are arranged between pinhole array disk 4 and sample 8 .
  • the micro-scope is designed so that the return light beam from sample 8 , that is reflected by beam splitter 3 , is made incident to CCD (Charge Coupled Device) camera 30 through relay lenses 29 .
  • CCD Charge Coupled Device
  • the embodiment operates as follows.
  • the laser light beam, introduced through FC connector 21 is made incident to collimating lens 23 , after changing its direction with mirror 22 , where the beam is converted to a parallel beam, and then is made incident to ND filter 24 , which reduces the light intensity thereof.
  • ND filter 24 After the light intensity has been reduced through ND filter 24 , the direction of the beam is changed by mirror 25 , and the beam is made incident to polarizer 2 .
  • the laser light beam is converted to a linearly polarized light beam by being passed through polarizer 2 .
  • the light beam in polarized state after having its direction of travel changed by mirror 26 , is focused with micro lenses in micro lens disk 27 and irradiates each. pinhole in pinhole array disk 4 corresponding to each micro lens after being transmitted through beam splitter 3 .
  • a light beam reflected from the surface of pin-hole array disk 4 reaches analyzer 9 after being reflected by beam splitter 3 .
  • the azimuthal angles of polarizer 2 and analyzer 9 are adjusted to attain the extinguished state, the light beam reflected from pinhole array disk 4 is cut off by analyzer 9 and does not reach CCD camera 30 , the extinction ratio being in the order of about 10 ⁇ 6 .
  • Tube lens 28 and objective lens 7 are constructed with a plurality of lenses and each lens causes a slight reflected light on its surfaces especially in the vicinity of the optical axis.
  • the reflected light beams reach analyzer 9 after passing through pinhole array disk 4 again and are reflected by polarized beam splitter 3 .
  • the reflected light beams do not reach CCD camera 30 since they are cut off by analyzer 9 for the same reason as for the pinhole array disk 4 .
  • the reflected light beams act as background light against the observation light, or as noise light.
  • the light beam that is focused on objective lens 7 is circularly polarized by the quarter wavelength plate 6 , which is disposed between objective lens 7 and sample 8 .
  • Circularly polarized spots are irradiated on sample 8 as approximately 1,000 very small spots.
  • the spots scan the surface of sample 8 forming a raster, each constituting a scanning line by rotation of the micro lens disk 27 and the pinhole array disk 4 .
  • the light beam focused on sample 8 is reflected by sample 8 and becomes a linearly polarized light beam again by being transmitted through the quarter wavelength plate 6 .
  • the light beam becomes a linearly polarized light beam whose azimuthal angle is inclined by 90°.
  • the light beam is focused by tube lens 28 and forms an angle on pinhole array disk 4 .
  • the light beam that passes through the pinholes is reflected by polarized beam splitter 3 , then is made incident on analyzer 9 , and then directly forms an image on CCD camera 30 through relay lens 29 .
  • observation of the image may be done by means other than the CCD camera 30 .
  • a CMOS image pickup camera or a photographing camera with film may be used.
  • the image can be directly observed through an eyepiece by the naked eye.
  • Polarizer 2 and analyzer 9 are arranged for the extinguished state.
  • the quarter wavelength plate 6 is placed at the azimuthal angle of 45° to the azimuth of the light beam irradiated from objective lens 7 , and is disposed between objective lens 7 and sample 8 .
  • the embodiment reduces “stray light” including reflected light generated by optical components located between the polarizer 2 and the quarter wavelength plate 6 , and between quarter wavelength plate 6 and analyzer 9 , by an extinction ratio of the order of 10 ⁇ 4 in light intensity.
  • the embodiment transmits light beam that is transmitted through plate 6 , that is to say, the reflected light from sample 8 is 100% transmitted as an observation light. This allows the image of the sample 8 to be observed clearly.
  • the quantity of light that reaches the CCD camera 30 was compared for the cases where an optical mirror was placed and was not placed in the position of the sample for each of the cases of the conventional microscope and the embodiment of the invention.
  • the optical mirror was used in place of sample 8 had a reflectivity of 36%.
  • the light intensity was 3.0 ⁇ W for the conventional microscope, and 0.090 ⁇ W for the invention microscope. This is equivalent to the background light or “noise light” generated inside the optical microscope.
  • the level of the noise light is fixed and the ratio of the above signal light to the noise light is the S/N ratio of the image obtained by the CCD camera.
  • the S/N ratio of the conventional microscope is 9.0 dB and that of the invention microscope is 23.6 dB.
  • transparent glass having the reflectivity of one order smaller (i.e. reflectivity of 3.6%) than the mirror having the reflectivity of 36% is used for the sample.
  • Observation cannot be done with a conventional microscope but can be done with the invention microscope, with a margin of one order of S/N ratio.
  • optical scanning is done by rotating a Nipkow disk in the above embodiment
  • scanning can be done by use of an ordinary mirror or by use of an acoustic optic element (AOM means Acoustic Optic Modulator).
  • AOM means Acoustic Optic Modulator
  • the placement of the quarter wavelength plate 6 is easy to accomplish if a sufficient gap , i.e. working distance, is provided between the objective lens 7 and sample 8 .
  • a sufficient gap i.e. working distance
  • placement of the quarter wavelength plate 6 is difficult because the working distance or gap is usually small.
  • an alternative arrangement such as shown in FIG. 3, would be to use a cover glass 41 placed on the surface of sample 8 , mounted on slide glass 42 , as the quarter wavelength plate.
  • Another alternative arrangement, as shown in FIG. 4 is to incorporate the quarter wavelength plate in an objective lens assembly 50 , and substitute the combination for the objective lens 7 .
  • two polarizers and a half mirror may be used in place of a polarized beam splitter 3 .
  • the invention has the following and other advanageous effects.
  • the S/N ratio is greatly improved and samples having low reflectivity can be measured by placing the quarter wavelength plate under the objective lens, that is on the sample side.
  • samples having low reflectivity can be measured by the invention with high S/N ratio even in an optical microscope using a confocal optical scanner that rotates a substrate having very small apertures.
  • samples having low reflectivity can also be measured by the invention at a high S/N ratio even in an optical microscope using a confocal optical scanner that performs optical scanning using a movable mirror or an acoustic optical modulator.
  • a polarized beam splitter or two polarizaers and a half mirror can be used as the beam spitter in the invention and obtain the advantage of increased flexibity of component selection. Furthermore, even if the distance between the objective lens and the sample is small, the quarter wavelength plate can be easily arranged in the invention between the objective lens and the sample. Also, advantageously, a Nipkow disk can be used in the invention as a substrate having a plurality of very small apertures. Finally and advantageously, in the invention, a light beam can be focused on each pinhole of the Nipkow disk for irradiation through micro lenses of a micro lens disk by mounting the micro lens disk in parallel with the Nipkow disk, which enhances the light utilization factor.

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  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Microscoopes, Condenser (AREA)
US09/257,154 1998-03-19 1999-02-24 Optical microscope Expired - Fee Related US6185035B1 (en)

Applications Claiming Priority (2)

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JP10-069649 1998-03-19
JP10069649A JPH11271622A (ja) 1998-03-19 1998-03-19 光学顕微鏡

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6974076B1 (en) * 2000-02-14 2005-12-13 Sony Corporation Portable music player with pay per play usage and method for purchase of credits for usage
WO2006117172A2 (de) * 2005-05-03 2006-11-09 Carl Zeiss Jena Gmbh Einrichtung und verfahren zur reproduzierbaren einstellung der pinholeöffnung und pinholelage in laserscanmikroskopen
US20070147673A1 (en) * 2005-07-01 2007-06-28 Aperio Techologies, Inc. System and Method for Single Optical Axis Multi-Detector Microscope Slide Scanner
US20100277794A1 (en) * 2009-04-30 2010-11-04 Olympus Corporation Microscope
US20110090223A1 (en) * 2004-05-27 2011-04-21 Aperio Technologies, Inc. Creating and viewing three dimensional virtual slides
JP2013011728A (ja) * 2011-06-29 2013-01-17 Yokogawa Electric Corp 顕微鏡装置
US8805050B2 (en) 2000-05-03 2014-08-12 Leica Biosystems Imaging, Inc. Optimizing virtual slide image quality

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4791690B2 (ja) * 2003-11-28 2011-10-12 株式会社ニコン 対物レンズ
KR100845284B1 (ko) * 2004-09-22 2008-07-09 삼성전자주식회사 두개의 닙코우 디스크를 이용한 공초점 주사 현미경
WO2007101205A2 (en) * 2006-02-27 2007-09-07 Aperio Technologies, Inc System and method for single optical axis multi-detector microscope slide scanner
JP5403404B2 (ja) * 2009-03-31 2014-01-29 株式会社ニコン 顕微鏡装置

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US5032720A (en) * 1988-04-21 1991-07-16 White John G Confocal imaging system
US5035476A (en) * 1990-06-15 1991-07-30 Hamamatsu Photonics K.K. Confocal laser scanning transmission microscope
US5067805A (en) * 1990-02-27 1991-11-26 Prometrix Corporation Confocal scanning optical microscope
US5162941A (en) * 1991-07-23 1992-11-10 The Board Of Governors Of Wayne State University Confocal microscope
US5303082A (en) * 1991-02-28 1994-04-12 Olympus Optical Co., Ltd. Stereomicroscope including two pair of polarizers and a quarter wavelength plate
US5428475A (en) * 1991-10-31 1995-06-27 Yokogawa Electric Corporation Confocal optical scanner

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5032720A (en) * 1988-04-21 1991-07-16 White John G Confocal imaging system
US5067805A (en) * 1990-02-27 1991-11-26 Prometrix Corporation Confocal scanning optical microscope
US5035476A (en) * 1990-06-15 1991-07-30 Hamamatsu Photonics K.K. Confocal laser scanning transmission microscope
US5303082A (en) * 1991-02-28 1994-04-12 Olympus Optical Co., Ltd. Stereomicroscope including two pair of polarizers and a quarter wavelength plate
US5162941A (en) * 1991-07-23 1992-11-10 The Board Of Governors Of Wayne State University Confocal microscope
US5428475A (en) * 1991-10-31 1995-06-27 Yokogawa Electric Corporation Confocal optical scanner

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6974076B1 (en) * 2000-02-14 2005-12-13 Sony Corporation Portable music player with pay per play usage and method for purchase of credits for usage
US9535243B2 (en) 2000-05-03 2017-01-03 Leica Biosystems Imaging, Inc. Optimizing virtual slide image quality
US8805050B2 (en) 2000-05-03 2014-08-12 Leica Biosystems Imaging, Inc. Optimizing virtual slide image quality
US8565480B2 (en) 2004-05-27 2013-10-22 Leica Biosystems Imaging, Inc. Creating and viewing three dimensional virtual slides
US9069179B2 (en) 2004-05-27 2015-06-30 Leica Biosystems Imaging, Inc. Creating and viewing three dimensional virtual slides
US8923597B2 (en) 2004-05-27 2014-12-30 Leica Biosystems Imaging, Inc. Creating and viewing three dimensional virtual slides
US20110090223A1 (en) * 2004-05-27 2011-04-21 Aperio Technologies, Inc. Creating and viewing three dimensional virtual slides
EP2098892A1 (de) * 2005-05-03 2009-09-09 Carl-Zeiss Jena GmbH Einrichtung und Verfahren zur reproduzierbaren Einstellung der Pinholeöffnung und Pinholelage in Laserscanmikroskopen
US8759745B2 (en) 2005-05-03 2014-06-24 Carl Zeiss Microscopy Gmbh Device for the reproducible adjustment of a pin hole opening and pin hole position in laser scanning microscopes using a reflector
WO2006117172A3 (de) * 2005-05-03 2007-03-22 Zeiss Carl Jena Gmbh Einrichtung und verfahren zur reproduzierbaren einstellung der pinholeöffnung und pinholelage in laserscanmikroskopen
WO2006117172A2 (de) * 2005-05-03 2006-11-09 Carl Zeiss Jena Gmbh Einrichtung und verfahren zur reproduzierbaren einstellung der pinholeöffnung und pinholelage in laserscanmikroskopen
US8164622B2 (en) * 2005-07-01 2012-04-24 Aperio Technologies, Inc. System and method for single optical axis multi-detector microscope slide scanner
US20070147673A1 (en) * 2005-07-01 2007-06-28 Aperio Techologies, Inc. System and Method for Single Optical Axis Multi-Detector Microscope Slide Scanner
US9235041B2 (en) 2005-07-01 2016-01-12 Leica Biosystems Imaging, Inc. System and method for single optical axis multi-detector microscope slide scanner
US20100277794A1 (en) * 2009-04-30 2010-11-04 Olympus Corporation Microscope
JP2013011728A (ja) * 2011-06-29 2013-01-17 Yokogawa Electric Corp 顕微鏡装置

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